The present invention relates to novel methods of treating and preventing disease caused by absence or deficiency of the activity of enzymes belonging to the heme biosynthetic pathway. More specifically, the invention pertains to methods of alleviating the symptoms of certain porphyrias, notably acute intermittent porphyria including gene therapy, therapy with a combination of encymatically active substances and therapy with recombinant produced enzymes such as PBGD. In addition the invention relates to an expression plasmid and a linear DNA fragment for use in the production of rhPBGD.
Heme Biosynthetic Pathway
Heme is a vital molecule for life in all living higher animal species. Heme is involved in such important processes as oxygen transportation (haemoglobin), drug detoxification (cytochrome P450), and electron transfer for the generation of chemical energy (ATP) during oxidative phosphorylation in mitochondria.
Heme is synthesised in eight consecutive enzymatic steps starting with glycin and succinyl-CoA. Sassa S. 1996, Blood Review, 10, 53-58 shows a schematic drawing (FIG. 1 in the article) of the heme biosynthetic pathway indicating that the first enzymatic step (delta-aminolevulinic-synthetase) and the last three steps (coproporphyrinogen oxidase, protoprophyrinogen oxidase and ferrochelatase) are located in the mitochondrion whereas, the remaining are cytosolic enzymes.
Important regulation of the heme biosynthetic pathway is delivered by the end product of the metabolic pathway, namely heme, which exerts a negative inhibition on the first rate-limiting enzymatic step (conducted by delta-aminolevulinic-synthetase) in the heme biosynthetic pathway (Strand et al. 1970, Proc. Natl. Acad. Sci. 67, 1315-1320).
Deficiencies in the heme biosynthetic enzymes have been reported leading to a group of diseases collectively called porphyrias.
A defect in the third enzymatic step leads to acute intermittent porphyria, AIP.
Acute Intermittent Porphyria
Acute intermittent porphyria (AIP) is an autosomal dominant disorder in man caused by a defect (50% reduction of activity) of the third enzyme in the heme biosynthetic pathway, prophobilinogen deaminase, (also known as porphobilinogen ammonia-lyase (polymerizing)), E.C. 4.3.1.8. (Waldenstrxc3x6m 1937, J. Acta.Med. Scand. Suppl.82). In the following, this enzyme and the recombinant human form will be termed xe2x80x9cPBGDxe2x80x9d and xe2x80x9crhPBGDxe2x80x9d, respectively.
Clinical Manifestation of AIP
The reduction is enzymatic PBGD activity makes this enzyme the rate limiting step in the heme biosynthetic pathway, with a concomitant increase in urinary and serum levels of delta-aminolevulinic acid (ALA) and porphobilonogen (PBG).
The clinical manifestation of API involves abdominal pain and a variety of neuropsychiatric and circulatory dysfunctions. As a result of the enzymatic block, heme precursors such as PBG and ALA are excreted in excess amounts in the urine and stool. In acute attacks, high levels of PGB and ALA are also found in serum. These precursors are normally undetectable in serum in healthy individuals.
The neuropsychiatric disturbances observed in these patients are thought to be due to interference of the precursors with the nervous system or due to the lack of heme. For instance, ALA bears a close resemblance to the inhibitory neurotransmitter 4-aminobutyric acid (GABA) and has been suggested to be a neurotoxin. (Jeans J. et al. 1996, American J. of Medical Genetics. 65, 269-273).
Abdominal pain is the most frequent symptom in AIP patients and occurs in more than 90% during acute attacks, which will be followed rapidly by the development of peripheral neuropathy with weakness in proximal muscles, loss of pinprick sensation, and paraesthesia. Tachycardia, obstipation or diarrhoea may also be present. During acute attacks behavioral changes, confusion, seizures, respiratory paralysis, coma and hallucinations may be present.
Hypertension is also associated with AIP, with as high as 40% of patients showing sustained hypertension between attacks. An association between chronic renal failure (Yeung L. et al. 1983, Q J. Med 52, 92-98) and AIP as well as hepatocellular carcinoma. (Lithner F. et al. 1984, Acta.Med.Scand. 215, 271-274), has been reported.
The AIP is a lifelong disease, which usually becomes manifest in puberty.
Factors Precipitating Acute Attacks
Most precipitating factors exhibit an association with the first rate-limiting enzyme in the heme biosynthetic pathway through heme, the final product of the pathway. A lowering of the heme concentration will immediately increase the rate of ALA-synthetase. An overproduction of ALA then makes the partially deficient PBGD enzyme (50% activity) now rate-limiting with an accumulation of the heme precursors ALA and PBG. Drugs that induces cytochrome P450 such as barbiturates, estrogens, sulphonamides, progesterone, carbamyazepine, and phenytoin can all precipitate acute attacks. (Wetterberg L. 1976, In Doss M. Nowrocki P. eds. Prophyrias in Human Disease. Reports of the discussion. Matgurg an der Lahn, 191-202).
The clinical manifestation is more common in women, especially at time of menstruation. Endocrine factors such as synthetic estrogens and progesterone are known precipitating factors. A significant factor is also the lack of sufficient caloric intake. Hence, caloric supplementation during acute attacks reduces clinical symptoms. (Welland, F. H. et al. 1964, Metabolism, 13, 232).
Finally, various forms of stress including illness, infection, surgery and alcoholic excess have been shown to lead to precipitation of acute attacks. There are also cases of acute attacks where no precipitating factor can be identified.
Prevalence of AIP Prevalence of 0.21% has been reported (Tishler P. V. et al. 1985, Am.J.Psychiatry 142, 1430-1436), with as high a prevalence as 1 per 1500 in geographic isolates in northern Sweden (Wetterberg L. 1967, Svenska bokfxc3x6laget Nordstedt, Stockholm). Prevalence up to 200 per 10,000 inhabitants has been reported from Arjepong in Northern Sweden (Andersson, Christer, Thesis, 1997, ISBN 91/7191/280/0, pp. 22-23).
Existing Treatment of AIP
The treatment of AIP as well as of other types of porphyrias such as variegata, hereditary coproporphyria, harderoporphyria, and aminolevulinic acid dehydratase deficiency, are basically the same. Existing therapies for AIP, are all aimed at reducing circulating PBG and ALA by inhibiting the first rate-limiting enzymatic step ALA-synthetase. This inhibition of ALA-synthetase is achieved by increasing circulating heme, since heme is a negative feed back regulator of ALA-synthetase. Hematin treatment, high caloric intake or inhibition of heme breakdown by Sn-mesoporphyrin administration are the existing therapies today. These therapies have shown limited efficacy.
Treatment between acute attacks involves sufficient caloric intake and avoidance of drugs and immediate treatment of infections.
Patients that experience acute attacks are treated with intravenous carbohydrates usually dextrose (300 g/day) and intravenous hematin (3-8 mg/(kg day)).
Treatments with long acting agonistic analogues of LHRH, have been shown to reduce the incidence of pre-menstrual attacks by inhibiting ovulation in AIP patients. Finally, treatments involving heme analogous Sn-mesoporphyrin, which inhibit heme breakdown have also been attempted.
Medical Need in AIP
The lack of effective treatment for AIP is well recognized. In a US mortality study in AIP patients requiring hospitalization it was concluded that the mortality rate was 3.2-fold higher as compared to a matched general population. Suicide was also a major cause of death, occurring at a rate of 370 times that expected in the general population (Jeans J. et al. 1996, Am. J. of Medical Genetics 65, 269-273).
Hematin therapy is usually initiated when high caloric intake is not sufficient to alleviate acute attacks. Studies with hematin have been performed but these studies generally used the patients as their own control after the patients did not respond to high carbohydrate treatment (Mustajoki et al. 1989, Sem. Hematol. 26, 1-9).
The one controlled study with hematin treatment reported, failed to reach statistical significance due to too small a patient number (Herric A. L. et al 1989, Lancet 1, 1295-1297).
In conclusion, there is a definite need for the provision of novel therapeutic/prophylactic methods aimed at these disease.
Levels of ALA and PBG found in urine in patients with symptomatic AIP, are in the range of 1-203 mg/day and 4-782 mg/day, respectively. Normal excretion of ALA and PBG is very low (0-4 mg/day). Important is the observation that these patients also have elevated levels of ALA and PBG in serum. It was shown in a study that AIP patients had significantly elevated levels of ALA (96 xcexcg %) and PBG (334 xcexcg %) in serum in connection with acute attacks and that the severity of the attacks were correlated to high levels of ALA and PBG. Hence, it is important to reduce the circulating levels of ALA and PBG in order to eliminate clinical symptoms and to normalize the heme pool.
The present inventors present a new therapeutic rational in the treatment of AIP, a rationale using PBGD, preferably recombinant human PBGD (rhPBGD), in order to reduce circulating high levels of PBG in serum by metabolizing (by enzymatic conversion) PBG to hydroxymethylbilane (HMB), which is the normal product of the reaction. This substitution therapy will lead to a normalization of PBG in serum as well as to a normalization of the heme pool. It will also lead to a normalization of ALA in serum, since these heme precursors are in equilibrium with each other. A lowering of serum ALA and PBG is expected to result in a concomitant relief of symptoms. The product of the reaction (HMB) will diffuse back into the cells and enter the normal heme biosynthetic pathway and will become subsequently metabolized to heme.
Alternatively investigations in treating the porphyrias have also suggested gene therapy, thus aiming at introducing genetic material in relevant cells, which will then take over the in vivo production of the enzyme of interest.
Hence, PBGD administered by injections will carry out its normal catalytic function by converting PBG to HMB in serum (extracellulary, not inside the cells). The new therapeutic idea is based on the assumption that ALA, PBG and HMB permeate cellular membranes or is transported specifically across them. An alternative to this is to administer a form of PBGD, which will be able to act intracellulary, either as a consequence of formulation or as consequence of modification of PBGD so as to facilitate its entry into cells from the extracellular compartment.
The observation that AIP patients have large amounts of these heme precursors in the serum supports the idea that PBG does not accumulate intracellularly, but is released from the cells into serum when the intracellular concentration increases due to the PBGD enzymatic block.
The basic new therapeutic concept for AIP is valid for all porphyrias and therefore the invention is in general aimed at treating these diseases by substituting the reduced or missing enzymatic activity characterizing the porphyrias.
Hence, in its broadest aspect, the invention pertains to a method for treatment or prophylaxis of disease caused by deficiency, in a subject, of an enzyme belonging to the heme biosynthetic pathway, the method comprising administering, to the subject, an effective amount of a catalyst which is said enzyme or an enzymatically equivalent part or analogue thereof.
Hence, by the term xe2x80x9ccatalystxe2x80x9d is herein meant either the relevant enzyme which is substituted as it is, or an enzymatically equivalent part or analogue thereof. One example of an enzymatically equivalent part of the enzyme could be a domain or subsequence of the enzyme which includes the necessary catalytic site to enable the domain or subsequence to exert substantially the same enzymatic activity as the full-length enzyme or alternatively a gene coding for the catalyst.
An example of an enzymatically equivalent analogue of the enzyme could be a fusion protein which includes the catalytic site of the enzyme in a functional form, but it can also be a homologous variant of the enzyme derived from another species. Also, completely synthetic molecules that mimic the specific enzymatic activity of the relevant enzyme would also constitute xe2x80x9cenzymatic equivalent analoguesxe2x80x9d.
In essence, the inventive concept is based on the novel idea of substituting the reduced enzymatic activity in the subject simply by administering a catalyst which will xe2x80x9cassistxe2x80x9d the enzyme which is in deficit. The precise nature, however, of the catalyst is not all-important. What is important is merely that the catalyst can mimic the enzymatic in vivo activity of the enzyme.
The term xe2x80x9cthe heme biosynthetic pathwayxe2x80x9d refers to the well-known enzymatic steps (cf. e.g. Sassa S. 1996, Blood Review, 10, 53-58) which leads from glycin and succinyl-CoA to heme, and enzymes belonging to this synthetic pathway are delta-aminolevulininic acid synthetase, delta-aminolevulinic acid dehydratase, porphobilinogen deaminase, uroporphyrinogen III cosythetase, uroporphyrinogen decarboxylase, coproporphyrinogen oxidase, protoporphyrinogen oxidase and ferrochelatase. Hence, in line with the above, a catalyst used according to the invention is such an enzyme or an enzymatically equivalent part or analogue thereof. It should be noted that the genes encoding all of the above-mentioned enzymes have been sequenced, thus allowing recombinant or synthetic production thereof.
The diseases related to reduced activity of these enzymes are acute intermittent porphyria (AIP), ALA deficiency porphyria (ADP), Porphyria cutanea tarda (PCT), Hereditary coproporphyria (HCP), Harderoporphyria (HDP), Variegata porphyria (VP), Congenital erthropoietic porphyria (CEP), Erythropoietic protoporphyria (EPP), and Hepatoerythropoietic porphyria (HEP).
By the term xe2x80x9ceffective amountxe2x80x9d is herein meant a dosage of the catalyst which will supplement the lack or deficiency of enzymatic activity in a subject suffering from porphyria caused by reduced activity of one of the above-mentioned enzymes. The precise dosage constituting an effective amount will depend on a number of factors such as serum half-like of the catalyst, specific activity of the catalyst etc. but the skilled person will be able to determine the correct dosage in a given case by means of standard methods (for instance starting out with experiments in a suitable animal model such as with transgenic animals so as to determine the correlation between blood concentration and enzymatic activity).
The disease which is the preferred target for the inventive method is AIP, and therefore the catalyst is PBGD or an enzymatically equivalent part or analogue thereof. It is most preferred that the catalyst is a recombinant form of the enzyme belonging to the heme biosynthetic pathway or of the enzymatically equivalent part or analogue thereof, since recombinant production will allow large-scale production which, with the present means available, does not seem feasible if the enzyme would have to be purified from a native source.
Preferred formulations and dosage forms of the catalyst are exemplified for, but not limited to, PBGD in the detailed description hereinafter, and these formulations also are apparent from the claims. It will be appreciated that these formulations and dosage forms are applicable for all catalysts used according to the invention.
One important embodiment of the method of the inventions in one wherein the catalyst, upon administration, exerts at least part of its enzymatic activity in the intracellular compartment. This can e.g. be achieved when the catalyst is an enzymatically equivalent part or analogue of the enzyme, since such variations of the enzyme can be tailored to render them permeate cell membranes. Hence, when the catalyst is a small artificial enzyme or an organic catalyst which can polymerize porphobilinogen to hydroxymethylbilane, it should be possible for the skilled man to introduce relevant side chains which facilitates entry into the intracellular compartment. Alternatively, the catalyst is the enzyme, but formulated in such a manner that it exerts at least part of its enzymatic activity intracellularly upon administration to the subject. This can be achieved by tagging the enzyme with specific carbohydrates or other liver cell specific structures for specific liver uptake, i.e. the enzyme (or analogue) is modified so as to facilitate active transport into e.g. liver cells.
Although the above embodiments are interesting, it is believed that the normal, practical embodiment of the invention will involve use of a catalyst which exerts substantially all its enzymatic activity extracellularly in the bloodstream, since it is believed that the metabolic products of the enzymatic conversion of the relevant heme precursor will permeate freely into the intracellular compartment where the remaining conversions of the heme biosynthetic pathway can take place. Alternatively, the metabolic product may be excreted from the subject via urine and/or faeces at least to some extent.
As mentioned above, it is preferred that the catalyst is produced recombinantly, i.e. by a method comprising
a) introducing, into a suitable vector, a nucleic acid fragment which includes a nucleic acid sequence encoding the catalyst;
b) transforming a compatible host cell with the vector;
c) culturing the transformed host cell under conditions facilitating expression of the nucleic acid sequence; and
d) recovering the expression product from the culture and optionally subjecting the expression product to post-translational processing, such as in vitro protein refolding, enzymatic removal of fusion partners, alkylation of amino acid residues, and deglycosylation, so as to obtain the catalyst.
For relatively small catalysts (e.g. those constituted mainly of the active site of the enzyme), the catalyst can alternatively be prepared by liquid-phase or solid-phase peptide synthesis.
A more detailed explanation of the recombinant production of the model enzyme PBGD is given in the detailed section hereinafter, but as mentioned herein the same considerations apply for all other peptide catalysts of the invention. One of the main advantages of producing the catalyst by recombinant or synthetic means is, that if produced in a non-human cell, the catalyst is free from any other biological material of human origin, thus reducing problems with known or unknown pathogens such as viruses etc.
The dosage regiment will normally be comprised of at least one daily dose of the catalyst, (preferably by the intravenous route). Normally 2, 3, 4 or 5 daily dosages will be necessary, but if sustained release compositions are employed, less than 1 daily dosage are anticipated.
The daily dosage should be determined on a case by case basis by the skilled practitioner, but as a general rule, the daily dosage will be in the range between 0.01-1.0 mg/kg body weight per day of the catalyst. More often the dosage will be in the range of 0.05-0.5 mg/kg body weight per day, but is should never be forgotten that precise dosage depends on the dosage form and on the activity of the catalyst as well as on the degree of deficiency of the relevant enzyme or combinations of enzymes and an individualized treatment, where the dose is adjusted to normalize patient serum and urine precursor levels.
The most correct way of determining the correct dosage is based on the patient specific precursor levels. The precursor being the product of the enzymatic reaction.
For PBGD, the daily dosage is about 0.08-0.2 mg per kg body weight per day, and most often 0.1 mg per kg body weight per day will be the dosage of choice. It is believed that comparable dosages will be applicable for the other full-length enzymes or combinations of enzymes.
Finally, as will be appreciated from the above disclosure, the invention is based on the novel idea of providing substitution for the enzymes lacking in activity. To the best of the knowledge of the inventors, therapeutic use of catalysts having such effects have never been suggested before, and therefore the invention also pertains to a catalyst as defined herein for use as a pharmaceutical. Furthermore, use of such catalysts or combination of different catalysts for the preparation of pharmaceutical compositions for treatment of the above-discussed disease is also part of the invention.